Morphological Study of the Gastrointestinal Tract of Larimichthys

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ZOOLOGIA 35: e25171 ISSN 1984-4689 (online)

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RESEARCH ARTICLE Morphological study of the gastrointestinal tract of Larimichthys crocea (Acanthopterygii: Perciformes)

Hameeda Kalhoro 1,2, Shengli Tong 1, Lei Wang 1, Ying Hua 1, Josie Ancella Volatiana 1, Qingjun Shao 1

1Aquaculture Nutrition Laboratory, College of Animal Sciences, Zhejiang University. Hangzhou 310058, China. 2Department of Fresh Water Biology and Fisheries, Faculty of Natural Sciences, University of Sindh Jamshoro. Jamshoro 76080, Pakistan. Corresponding author: Qingjun Shao ([email protected]) http://zoobank.org/E6F123A8-B168-4AB7-A45F-F0FD375051A1

ABSTRACT. The present study aimed to investigate the macroscopic and histological structure of the gastrointestinal tract (GIT) of Larimichthys crocea (Richardson, 1846). It consists of esophagus, stomach regions, pyloric caeca, intestinal regions, and rectum. Sixteen tubular light yellowish pyloric caeca of similar sizes were observed in all individuals. The digestive wall consists of mucosa, submucosa, muscularis, and adventitia. No major differences appeared in the structure of the tunica, epithelial cell types, connective tissues and musculature glands of L. crocea GIT. The mucosal epithelia in the oesophagus has longitudinal branched folds with frontward and hindmost zones. The gastric tunica mucosa has a characteristic folded structure and can be divided into three regions. The intestinal tunica mucosa is characterized by villi structures and numerous mucus-secreting cells. Mucus-secreting goblet cells were strongly positive to AB at pH 2.5 in the oesophagus (excluding gastro-oesophageal junction) and intestine mucosal regions, which indicates an abundance of carboxylate mucins. The surface epithelia of the gastric mucosa is PAS-positive and AB-negative. SEM examination revealed that cells in the epithelium of the esophagus have an unbroken apical layer and goblet cells. The intestinal coefficient (IC) of L. crocea was 0.80 ± 0.21, consistent with a carnivorous or omnivorous habit. Our study adds the knowledge of the digestive system of L. crocea and might be useful in the management of L. crocea stocks. KEY WORDS. Anatomy, epithelial mucins, gastrointestinal tract, intestinal coefficient, large yellow croaker.

INTRODUCTION The large yellow croaker, Larimichthys crocea (Richardson, 1846), known as drums or croakers in reference to the repetitive Morphological studies help in understanding the rela- drumming echoes they make, belong to Sciaenidae (Ramcharitar tionship between physiological and biochemical functions et al. 2006). This important carnivorous marine fish species is and molecular mechanisms. The gastrointestinal tract (GIT) cultivated in many Asian countries, particularly China (Feng of fish has shown a remarkable diversity of morphological and Cao 1979). The ontogenetic development of the digestive and functional traits, particularly in connection with unusual tract of L. crocea was briefly described by Mai et al. (2005). nutritional behaviors, body shape and sex (Díaz et al. 2008b, Young fish start out with a simple, indistinguishable GIT that Kapoor et al. 1975). There have been many contributions on changes during development according to the environmental the morphology of the GIT structure of fish, including marine, conditions and prey availability (Govoni et al. 1986). Function- freshwater and estuarine species (Murray et al. 1994, Park and al differences in the GIT of fish are reflected in their capacity Kim 2001). Furthermore, due to various structural differences to digest, absorb and assimilate chemical compounds and are in the GIT of fish, nutritional studies have been the focus of also associated with the developmental situation (Dabrowski most research in the field. For instance, past research has tried 1984). In aquaculture, studying the anatomical and histological to correlate morpho functional characters of the GIT of fish with characteristics of the GIT of fish may help to understand the their nutritional habits and behaviors, the results of which are development of pathological conditions, to devise nutritional important to commercial fish management. formulations and promote the management of L. crocea stocks

ZOOLOGIA 35: e25171 | DOI: 10.3897/zoologia.35.e25171 | November 1, 2018 1 / 9 H. Kalhoro et al. in aquaculture. The primary goal of the present study was to ± 1.01 cm), mean standard length (22.78 ± 1.00 cm), intestinal describe the macroscopic and histological organization of the length (18.29 ± 4.76 cm) and intestinal coefficient (0.80 ± 0.21 GIT of L. crocea. IC). The GIT of L. crocea consists of the esophagus, stomach regions, pyloric caeca, intestinal regions, and rectum. The short MATERIAL AND METHODS esophagus connects the pharyngeal cavity with the stomach. The u-shaped stomach is a muscular sack between the esophagus and Twelve mature L. crocea were obtained from the Marine the anterior intestine and sits behind the liver. Sixteen tubular Fisheries Research Institute of Zhejiang Province, Zhoushan, light yellowish pyloric caeca of similar sizes were observed in all China. After being anesthetized with tricaine methanesulfonate, individuals. The short intestine extends from the pyloric region MS 222; Sigma Chemical Co. solution by using a dosage of 100 of the stomach to the rectum and can be divided into anterior, mg L−1, the fish were euthanized via a longitudinal incision middle and posterior regions (Fig. 1). through their ventral side. The length of the intestine was measured to the nearest millimeter, using a tape measure and the intestinal coefficient (IC) was calculated using the equation IC = IL/SL (Angelescu and Gneri 1949). The segmental tissue fragments were removed after dissection and immediately processed for histological, histochemical and scanning electron microscopic studies. Sliced tissue fragments from the separated GIT were fixed in 10% formalin (for 24 hours). After fixation, tissues were repeatedly washed in 70% ethanol and dehydrated via ethanol series. Then tissues were cleared in xylene and em- bedded using paraffin wax at 56–58 °C. Sections were sliced at 5 µm using a 820 HistoStat Reichert Rotatory Microtome (Reichert Technologies, NY, USA). After routine histological procedure, dewaxed sections were stained with haematoxy- lin – eosin (HE) solutions. Stained slides were examined and photographed under Olympus B202 microscope (Olympus Optical, Ltd, Tokyo, Japan). The fragments were preserved and processed similarly to Figure 1. Photograph of the L. crocea gut illustrating: (ES) esophagus, the procedure described above. Five to seven µm sections were (CS) cardiac stomach, (FS) fundic stomach, (PS) pyloric stomach, prepared and stained in Periodic acid-Schiff (PAS), Alcian blue (PC) pyloric caeca, (AI) anterior intestine, (MI) mid intestine, (PI) (AB pH 2.5) and PAS+AB pH 2.5 to identify the neutral and acidic posterior intestine, and (RE) rectum. Scale bar = 7 cm. mucins respectively. The sample processing method for scanning electron microscopy is described elsewhere in detail (Kalhoro et al. 2017). Histology and scanning electron microscopy (SEM) In brief, tissues were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in phosphate buffer (pH 7.0) (for 24 hour at 4 The digestive wall consists of mucosa, submucosa, muscu- °C). After fixation, tissues were post-fixed for 1–2 hours in 1% laris, and adventitia. No major differences were observed at the osmium tetroxide (pH 7.0) and desiccated in a graded ethanol structure of the tunic structure, epithelial cell types, connective sequence placed in a mixture of alcohol and isoamyl acetate (v: tissues and musculature glands in the GIT of L. crocea with v = 1:1) about 30 minutes, then shifted to pure isoamyl acetate respect to other observed individuals. The tunica muscularis for overnight. Finally, tissues were washed and desiccated in of the esophagus appears rigid, forming two muscular layers, inner longitudinal layer, and outer circular layer. The tunica Hitachi Model HCP – C critical-point dryer through liquid CO2. Desiccated samples were coated with gold-palladium in Hitachi muscularis in the stomach is also rigid and forms two smooth Model E-1010 ion sputter for 4–5 minutes and observed in SEM muscular layers, inner circular layer, and outer longitudinal layer. (TM-1000, Hitachi, Japan). The mucosal surface in the proximal intestine has numerous elongated and deep folds lined by simple columnar goblet cells. In the distal intestine, the folds are fewer and shorter, contain- RESULTS ing large numbers of goblet cells, and no glands in the mucosa Gross anatomy and submucosa. The absence of muscularis mucosa prevented a distinct separation between the lamina propria and the sub- The body measurements obtained to L. crocea (n = 12) mucosa. The tunica muscularis in the intestine is organized in were: body weight (198.79 ± 20.36 g), total body length (25.83

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Figures 6–8. Photograph illustrating the transverse section of oeso phagus containing mucus cells (arrow), lamina propria and lumen Figures 2–5. Photograph of L. crocea GIT illustrating the general (6–7 stained with Hematoxylin and eosin stain). (8) Mucus-secreting organization: (2) transversal section of the oesophagus, note the cells in the anterior part of the oesophagus (arrows) possessing muscular possessing an inner longitudinal layer and an outer circular neutral and acidic carboxylated and sulphoglycoprotein. Combined layer; (3) transversal section of stomach showing the muscularis stain of Periodic acid-Schiff + Alcian blue pH 2.5. (MC) Mucus cells, mucosa; (4) anterior intestine possessing longitudinal villi and thin (LP) lamina propria, (LU) lumen, (GC) goblet cells. muscular layer; (5) posterior intestine possessing short villi and thick muscular layer. Hematoxylin and eosin stain. (TM) Tunica mucosa, 26). Apical epithelia lined with a monolayer of goblet cells in (TS) tunica submucosa, (CL) circular layer, (LL) longitudinal layer, increased concentrations, goblet cells filled with secretory granu (SE) serosa. les and occupying most of the cytoplasm. There is a definite change in the epithelium of the mucosa two smooth muscle layers; inner circular and outer longitudinal at the gastro-esophageal junction. The boundary between these layers (Figs 2–5). two areas is conspicuous, with short non-glandular mucosal The esophageal tunica mucosa has deeper longitudinal area at esophageal-gastric connection and a transition from branched folds. Frontward or anterior and hindmost or poste- squamous epithelium with goblet cells in the esophagus to a rior regions in the epithelium are present. Stratified squamous simpler columnar epithelium lacking goblet cells in the gastric cells are present in the frontward zone, with an increased region (Figs 9, 10). The striated muscle fibers of the esophagus concentration of slightly undifferentiated multilateral mucous are progressively replaced with smooth muscle in the cardiac secreting cells surmounting a deep basal cell layer that opens stomach, and the circular muscle layer is internal to the new towards the esophageal lumen (Figs 6, 7). Squamous stratified longitudinal muscle layer (Fig. 11). epithelia are eventually replaced, in the hindmost zone, by Similar to the esophagus, the gastric mucosa has distinc- simple columnar epithelia, performing secretive functions. The tive folds. The mucosa is lined with columnar epithelia and stratum compactum is surrounded by densely packed connective can be divided into three regions: cardiac, fundic and pyloric tissue fibers and detaches the tunica mucosa from the tunica (Figs 13–15). The lamina propria and stratum compactum are submucosa. The tunica submucosa is made of loose connective observable. Gastric pits have a gland opening from the basal tissue. Stratification of the outermost covering tunica adventitia part that appear entirely distinct in the pyloric region. Glands irregular, as in other isolated parts of the mesothelium with and mucus-secreting cells predominate within the cardiac and slight underlying loose connective tissue. In other places, the fundic parts (Fig. 16). SEM confirmed the tall mucosal folds of tunica adventitia underlines with a thick connective tissue layer the stomach within the distal part and the presence of cardiac where blood vessels fold, extending from the esophageal wall, as well as fundic glands (Figs 27, 28). and containing large blood vessels. The mucosal structure of the pyloric caeca is formed by Under the SEM, cells in the epithelium of the esophagus lengthy thin bends, lined with simple columnar epithelia, rare are visible on an unbroken apical layer along with goblet cells mucus-secreting goblet cells and loose connective tissue within (Fig. 25). Aperture of Goblet cells revealing expelled mucus (Fig. lamina propria and small blood vessels (Fig. 17). Epithelia with

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Figures 17–18. Photograph illustrating the transverse section of pyloric caeca that histologically resembles with intestine, but with exception of the higher quantity of goblet cells (arrows), (17) stained with Hematoxylin and eosin stain and (18) stained with the combined stain of Periodic acid-Schiff + Alcian blue pH 2.5. (LU) Lumen, (GC) goblet cells.

Figures 9–12. Photograph illustrating the changing of oesophageal epithelium to the gastric epithelium (arrow) in (9) and (10). Note the absorptive cells are responsible for nutrient absorption. No short non-glandular mucosal region of the gastric epithelium, (11) tunica submucosa is present. In its place there is a thin circular replacement of striated muscle by smooth muscle and (12) rapid layer of smooth muscle. transform of oesophageal epithelia with goblet cells (arrow) to gas- Though no macroscopic differences can be observed in tric epithelia with epithelial cells containing apical mucosubstances. the intestine, in histological terms it is divided into anterior, (9–11) stained with Hematoxylin and eosin stain and (12) stained middle and posterior intestines (Figs 19–21). The tunica muco- with the combined stain of Periodic acid-Schiff + Alcian blue pH sa is characterized by the presence of single-layered epithelia, 2.5. (LP) Lamina propria, (TS) tunica submucosa, (GP) gastric pits. mostly consisting of enterocytes, mucus-secreting epithelium cells and no propria glands. Intraepithelial lymphocytes (IEL) are also observed in the epithelia. The superficial epithelial layer of all regions is covered by microvilli (brush border). The underlying connective tissue is scattered, containing blood vessels and nerves. Since the mucosal muscular thin layer is absent, the muscular mucosal thin layer and tunica submucosa are inconspicuous. The SEM observation on the anterior intestine shows the organization of longitudinal mucosal folds with visible intestinal crypts (Fig. 29). Distally along the intestinal segments, the high mucosal folds appear loosely organized and have asym- metrical structure. The folds decrease in size distally along the intestinal tube. Microvilli and the goblet cells opening towards luminal surface are visible on the posterior intestine (Fig. 30).

Mucin histochemistry

Histochemical analysis of epithelial mucins in the L. crocea GIT revealed secretory cells and goblet cells in the esophagus and intestine, and epithelial cells in the stomach. Mucus-secreting goblet cells were strongly positive to AB at pH Figures 13–16. Photograph illustrating the cardiac, fundic and 2.5 in the esophagus (excluding gastro-esophageal junction) pyloric regions in (13), (14), and (15) respectively stained with and the mucosal regions of the intestine have great amounts Hematoxylin and eosin stain and (16) stained with the combined of carboxylated mucins. The surface epithelia of the gastric stain of Periodic acid-Schiff + Alcian blue pH 2.5. (TM) Tunica mu- mucosa were PAS-positive and AB-negative (Fig. 16); a gel-like cosa, (GP) gastric pits, (CG) cardiac gland, (FG) fundic gland, (LP) structure is formed by acidic mucins covering the epithelia of lamina propria, (MC) mucus-secreting cells. the esophagus (Fig. 8). The pyloric caeca are positive for PAS

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Figures 19–24. Photograph illustrating the anterior, mid and posterior regions in (19, 22), (20, 23) and (21, 24), respectively. (19–21) stained with Hematoxylin and eosin stain and (22–24) stained with the combined stain of Periodic acid-Schiff + Alcian blue pH 2.5. (GC) Goblet cells, (MV) microvilli.

Figures 25–30. Scanning electron microscopy illustrating (25) epithelial surface of the oesophagus (26) pores of goblet cells (27) epithelial surface of the cardiac stomach showing cardiac gland (28) fundic region of the stomach showing fundic glands (29) Cross-section of the anterior intestine showing longitudinal mucosal folds, crypts, propria-submucosa and tunica muscularis (30) posterior intestine showing microvilli and pores of goblet cell. (M) Mucosa, (MF) mucosal fold, (PG) pores of goblet cells, (CG) cardiac gland, (PS) propria-submucosa, (FG) fundic gland, (CY) crypts, (MV) microvilli.

ZOOLOGIA 35: e25171 | DOI: 10.3897/zoologia.35.e25171 | November 1, 2018 5 / 9 H. Kalhoro et al. and negative for AB at pH 2.5, indicating neutral glycoproteins 1974). The formation of striated skeletal muscle fibers present in secretion (Fig. 18). The combined reaction of PAS + AB pH 2.5 the esophageal wall found in L. crocea has also been observed in showed a bluish staining of intestinal mucus-secreting goblet Stegastes fuscus (Cuvier, 1830), Pterodoras granulosus (Valenciennes, cells (Figs 22–24). The epithelium of the esophagus is unstained 1821) and Oligosarcus hepsetus (Cuvier, 1829) (Canan et al. 2012, in the combined reaction of PAS + AB pH 2.5. Moreover, the Germano et al. 2014, Vieira-Lopes et al. 2013). It provides expedi- oxynticopeptic cells of gastric glands reacted positively to the tious mobility with powerful contractions to clear the esophageal PAS reaction (Fig. 12) (Table 1). lumen (Chaves and Vazzoler 1984). As observed in our study, the presence of muscular layers in the esophagus (longitudinal inner DISCUSSION sublayer and circular outer sub layer) and striated muscles that may possibly expand the luminal surface area of the esophagus, Morphological studies on the GIT of fish are useful when enable food intake and have been similarly observed in other developing strategies for the management and conservation of Sciaenidae species such as Micropogonias furnieri (Desmarest, 1823) fish species (Germano et al. 2014, Xiong et al. 2011). Our results (Diaz et al. 2008a, b) and other teleosts; Hemisorubim platyrhyn- revealed some peculiarities of the GIT of L. crocea, including the chos (Valenciennes, 1840) (Faccioli et al. 2014) and Odontesthes rather uniform structure of a shorter esophagus, well-developed bonariensis (Valenciennes, 1835) (Diaz et al. 2006). In many stomach, pyloric appendages, and intestine. teleostean families, the stomach is absent (Genten et al. 2009). In teleosts, the length of the intestine is correlated with However, it is developed in Sciaenidae (Andrade et al. 2017). The nutritional habits (Canan et al. 2012, Machado et al. 2013). On stomach of L. crocea has a U-shaped saccular structure, as report- the basis of the IC obtained (0.80), L. crocea can be categorized as ed by Pessoa et al. (2013) for Hypostomus pusarum (Starks, 1913) omnivorous or carnivorous (0.2–2.5) (Bertin 1958), whereas the and by Andrade et al. (2017) in M. furnieri. A U-shaped stomach intestinal coefficient assortment for omnivorous (0.6–8.0) and fundus characterizes a debris-eater (Moraes et al. 1997), while a herbivorous (0.8–15.0) teleosts is far greater (Al-Hussaini 1949, Y-shaped, caecum sort of stomach is adapted to the assimilation Bertin 1958, Xiong et al. 2011). However, some herbivorous of the entire prey (Rodrigues et al. 2008). The gastric glands are teleosts have short intestines with less intestinal coefficients the main part of the gastric mucosa in vertebrates and teleost (Takesue 1954).Thus the intestinal coefficient might only be a species (Al Abdulhadi 2005). The epithelium of L. crocea contains criterion-referenced factor for classification of fish feeding habits. tubular gastric glands in cardiac and fundic regions, as reported in Similar to other teleosts, the esophageal mucosa of L. Sparus aurata Linnaeus, 1758, Pelteobagrus fulvidraco (Richardson, crocea has squamous epithelium (frontward zone) and columnar 1846), Glyptosternum maculatum (Regan, 1905) and Hemisorubim epithelium (hindmost zone) (Carrassón et al. 2006, Cataldi et al. platyrhynchos (Valenciennes, 1840) (Cao and Wang 2009, Cataldi 1987). Frontward, the mucous cells of the epithelium contain sia- et al. 1987, Faccioli et al. 2014, Xiong et al. 2011). Our results losulphon glycoproteins. This secretion increases in the frontward showing an absence of gastric glands within the pyloric part of zone and most likely protect the anterior region of the esophagus. the stomach confirm the results reported by (Clarke and Witcomb The histochemical staining reacted positively, confirming the 1980, Purushothaman et al. 2016). On the other hand, Arellano et function of mucus in regulating lubrication (Oliveira Ribeiro al. (2001) reported that gastric glands were absent from the cardiac and Fanta 2000). The lack of taste buds and lamina muscularis part of the stomach, while tubular glands within the pyloric part mucosae indicates that this organ has the function of pushing of the stomach were reported for a few teleosts by Al-Hussaini the swallowed micronutrients towards the gut (Ezeasor 1984). The (1949). The gastric mucosa of L. crocea consists epithelial cells submucosal part is a thick layer of connective tissue associated rich in neutral mucins similarly as reported for other teleosts with a thick muscle layer, which might be compensate for the (Cao and Wang 2009, Cataldi et al. 1987, Hernández García et al. absence of a mucosal muscular layer (Albrecht et al. 2001). In 2001), formed by microvilli that may possibly serve in nutritive fish, abundant mucous cells in the esophagus help food pass to absorption (Grau et al. 1992, Ortiz-Delgado et al. 2003). On the the stomach (Cataldi et al. 1987, Grau et al. 1992); those cells are other hand, this secretion from neutral mucous may protect equivalent to esophageal glands found in the lamina propria or epithelia from auto-digestion by hydrochloric acid and gastric in the submucosa of higher vertebrates (Andrew and Hickman glands secretion (Ferraris et al. 1987).

Table 1. Histochemical techniques of mucosubstances in epithelia of the Larimichthys crocea gastrointestinal tract.

Procedure Region Oesophagus Cardiac stomach Fundic stomach Pyloric stomach Pyloric caeca Anterior intestine Posterior intestine PAS ++ ++ ++ ++ + ++ ++ AB pH 2.5 – – – – ++ ++ ++ AB + PAS (pH 2.5) + + + + + ++ ++ Staining reaction: (–) negative; (+) medium; (++) strong.

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Pyloric caeca or tubular pouches vary in size and quantity the epithelial cells and mucus-producing cells indicate an active according to the nutritional habits of teleosts (Canan et al. 2012, role in the teleost gut immune response. Particularly, they are Purushothaman et al. 2016). Sixteen tubular pouches of almost involved in a regulatory function increasing the lymphocytic similar sizes were observed in L. crocea. A different amount has number in the intestinal mucosa, indicating the presence of a been found in different teleost such as 10 in Leporinus friderici mucosal immune system (Miura et al. 2012). (Bloch, 1794), 4 tubular pouches in Acanthopagrus schlegelii On the basis of our results, the morphology of the GIT of (Bleeker, 1854) and 5 in Lates calcarifer (Bloch, 1790) respectively L. crocea is consistent with omnivorous and carnivores feeding (Albrecht et al. 2001, Kalhoro et al. 2017, Purushothaman et al. habits. Further detailed studies should be conducted on the 2016). It is predicted that the number of intestinal caeca and ultrastructure level of the gastrointestinal tract of L. crocea. the length of the intestine are greater in herbivorous species, intermediate in omnivorous ones, and minimized in carnivorous ACKNOWLEDGEMENTS fish, but is not always consistent (Alonso et al. 2015). From a histological categorization, the tunica mucosa has simple prismatic This study was supported by Zhejiang Provincial Bureau of epithelia with mucus-secreting cells and longitudinal folded Science and Technology (Project 2015C02020, 2015C03010) and structure. The chief purpose of the tubular pouches is explained Zhejiang Aquaculture Nutrition & Feed Technology Service Team as “absorption” in contrast with mammals that function as fer- (ZJANFTST2017–1). We would like to thank the China-Norwegian mentation. These are filled up as well as collapsed together by Joint Laboratory of Nutrition and Feed for Marine Fish (Xixi Is- the anterior intestine, expanding the entire absorptive surface. land, Zhoushan, Zhejiang Province) for their technical assistance. The intestinal mucosa is covered by columnar epithelia (enterocytes) and dominant mucus cell type called the goblet LITERATURE CITED cell, like in other teleosts. Carrassón et al. (2006) stated that gel-forming mucin secretion produced by these goblet cells is Al-Hussaini AH (1949) On the functional morphology of the al- involved in epithelial protection as well as in lubrication for imentary tract of some fish in relation to differences in their nutrient pass way. The increased concentration of mucus-se- feeding habits; anatomy and histology. 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